A variety of different hosts including plants, algae, fungi, stramenopiles and yeast have been and continue to be investigated as means for commercial production of polyunsaturated fatty acids (PUFA). Genetic engineering has demonstrated that the natural abilities of some hosts, even those natively limited to linoleic acid (LA, 18:2 omega-6) or alpha-linolenic acid (ALA, 18:3 omega-3) fatty acid production, can be substantially altered to result in high-level production of various long-chain omega-3/omega-6 PUFAs.
Although the literature reports a number of recent examples whereby various portions of the omega-3/omega-6 PUFA biosynthetic pathway responsible for eicosapentaenoic acid (EPA) production have been introduced into plants and non-oleaginous yeast, significant efforts have focused on the use of the oleaginous yeast, Yarrowia lipolytica (U.S. Pat. Nos. 7,238,482 and 7,932,077; U.S. Pat. Appl. Publ. Nos. 2009-0093543 and 2010-0317072). Oleaginous yeast are defined as those yeast that are naturally capable of oil synthesis and accumulation, wherein oil accumulation is at least 25% of the cellular dry weight, or those yeast genetically engineered such that they become capable of oil synthesis and accumulation, wherein oil accumulation is at least 25% of the cellular dry weight.
Emphasis has been placed on the development of transgenic oleaginous Y. lipolytica strains that can produce enhanced amounts of EPA. This focus on EPA production is due in part to the recognized salutary effects of EPA. For example, EPA has been shown to play a role in maintaining brain, retina and cardiovascular health. EPA is also known to have anti-inflammatory properties and may be useful in treating or preventing diseases linked to inflammation, such as cardiovascular disease and arthritis. Thus, the clinical and pharmaceutical value of EPA is well known (U.S. Pat. Appl. Publ. No. 2009-0093543). Similarly, the advantages of producing EPA in microbes using recombinant means, as opposed to producing EPA from natural microbial sources or via isolation from fish oil and marine plankton, are also well recognized. Interest in EPA production in yeast has also been due to the drive to develop sustainable sources of EPA as alternatives to producing EPA from fish, which would help alleviate problems associated with overfishing.
Enhanced EPA production in Y. lipolytica has been targeted in two general ways. First, attempts have been made to increase the amount of EPA present in the oil produced by Y. lipolytica. Such oil, which may not necessarily constitute a large percentage of the dry cell weight of Y. lipolytica biomass, can be purified away from the biomass, then used in EPA dietary supplements and/or used for further concentration for pharmaceutical applications. Attempts have also been made to increase the amount of EPA in the dry cell weight of Y. lipolytica. This entails trying to (i) increase the level of oil in Y. lipolytica while also (ii) increasing the amount of EPA present in the oil. The resulting biomass can be used directly in feeding schemes to deliver a high quantity of EPA in the diet while side-stepping issues of oil purification. Of course, such biomass can also serve as a source of oil in EPA supplements and the oil can also be used for further concentration for pharmaceutical applications, requiring less biomass per unit of EPA produced compared to Y. lipolytica biomass containing a lower amount of oil.
U.S. Pat. Appl. Publ. No. 2010-0317072 discloses a transgenic Y. lipolytica strain that produces oil containing 61.8% by weight EPA of the total fatty acids of the oil. However, this strain contains 26.5% oil on a dry cell weight basis. So, while the EPA content in the oil is high (61.8%), the EPA content in the disclosed Y. lipolytica strain on a dry cell weight basis is lower at about 16.4%.
A transgenic Y. lipolytica strain is disclosed in U.S. Pat. Appl. Publ. No. 2012-0052537 that produces oil containing 58.7% by weight EPA of the total fatty acids of the oil. This strain contains 38.3% oil on a dry cell weight basis. So, while the EPA content in the oil is high (58.7%), the EPA content in the disclosed Y. lipolytica strain on a dry cell weight basis is lower at about 22.5%.
U.S. Pat. Appl. Publ. No. 2012-0052537 also discloses a transgenic Y. lipolytica strain that produces oil containing 48.3% by weight EPA of the total fatty acids of the oil. On a dry cell weight basis, this strain contains 56.2% oil and an EPA content of about 27.1%.
These disclosed examples indicate that as improvements are made in developing transgenic Y. lipolytica strains for enhanced EPA and/or oil production, an inverse correlation arises between the total amount of oil produced and the amount of EPA present in the total fatty acids of the oil. Strains engineered to produce higher amounts of oil on a dry cell basis generally have lower amounts of EPA as a percentage of the fatty acids in the oil.
Increases in the total amount of EPA produced on a dry cell weight basis have been realized, even though there has been an inverse relationship between oil production and the amount of EPA produced as a percentage of the total fatty acids in oil. Despite this achievement, there is still a need to develop Y. lipolytica strains that can produce greater total amounts of EPA. Achieving this goal will likely entail the development of new strain modifications that enhance the amount of EPA as a percentage of the total fatty acids in oil, while not compromising the total amount of oil produced by the strain.
Polynucleotide sequences encoding the Sou2 sorbitol utilization protein have been identified by others. Information regarding the function of this protein, however, appears to be limited. For example, Jami et al. (2010, Molecular & Cellular Proteomics 9:2729-2744) disclosed that a “probable” Sou2 protein was present in the extra-cellular fraction of the filamentous fungus Penicillium chrysogenum. The characterization of several amino acid sequences in online databases as Sou2 sorbitol utilization protein appears to be based on sequence homology only without the disclosure of functional studies. The amino acid sequence of the Sou2 protein in Candida albicans is about 72% identical to the amino acid sequence of C. albicans Sou1 protein, which has been disclosed by Janbon et al. (1998, Proc. Natl. Acad. Sci. U.S.A. 95:5150-5155) and Greenberg et al. (2005, Yeast 22:957-969) to be a sorbose reductase required for L-sorbose utilization. However, despite the homology between C. albicans Sou1 and −2 proteins, Janbon et al. disclosed that the Sou2 protein is not required for sorbose utilization. The roles of the Sou1 and Sou2 proteins in lipid metabolism, if any, are believed to be unknown.
Studies are disclosed herein detailing the development of Y. lipolytica strains that can produce more than 28% EPA as dry cell weight. The modifications used to generate such strains included down-regulating a gene encoding Sou2 sorbitol utilization protein.